High peroxidase catalytic activity of exfoliated few-layer graphene

CARBON

6 2 ( 2 0 1 3 ) 5 1 –6 0

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High peroxidase catalytic activity of exfoliated few-layer graphene Zhenbing Wang, Xincong Lv, Jian Weng

*

Department of Biomaterials, College of Materials, Xiamen University, Xiamen 361005, PR China

A R T I C L E I N F O

A B S T R A C T

Article history:

Few-layer graphene prepared from graphite exfoliated by chitosan has a preserved struc-

Received 9 April 2013

ture without oxidation or destruction of the sp2 character of the carbon plane and exhibits

Accepted 26 May 2013

a higher peroxidase catalytic activity than that of graphene oxide (GO) and its reduced

Available online 5 June 2013

form. The peroxidase catalytic activity of as-obtained few-layer graphene is 45 times higher than that of GO and 4 times higher than that of reduced GO with the same concentration of 30 lg mL1 and the detection limit of hydrogen peroxide is 10 nM. The excellently catalytic performance can be attributed to the fast electron transfer on the surface of few-layer graphene, which is further confirmed by electrochemical characterization. The as-prepared few-layer graphene has been used to determine hydrogen peroxide in three real water samples with satisfactory results.  2013 Elsevier Ltd. All rights reserved.

1.

Introduction

With an atomically thick sheet structure and high conductivity, graphene has attracted intense interest in the field of biomedicine and biotechnology [1–7]. Recently great attention has been focused on the preparation of graphene oxide (GO) and reduced graphene oxide (RGO) [8–10], which provides the potential of cost-effective, large-scale production of graphene-based materials [11]. However, these methods suffer from one significant disadvantage because of the structural defects resulted from the oxidation process which disrupts the band structure and completely degrades the electronic properties that make graphene unique [8,12]. While some of the defects can be removed by reduction, large defect populations still remain, which continue to disrupt the electronic performance. However, pristine graphene with high conductivity is required in many applications. Research concentrated on exfoliating pristine graphite into graphene or few-layer graphene directly in organic solvent or aqueous solution with the aid of additives has been carried out [13,14]. However, the residual solvent or additives have a

bad effect on the property of graphene, which make them unavailable in practical application. Exfoliating graphite into few-layer graphene with chitosan directly in the aqueous solution has not been reported to the best of our knowledge. Chitosan, a renewable resource and the second abundant polysaccharide present in nature with good biocompatibility and biodegradability [15], has been shown to be a good polymeric dispersant for carbon nanotubes [16–18]. Enlightened by these works, here we report that few-layer graphene can be prepared from pristine graphite in aqueous solution with the aid of chitosan. X-ray photoelectron spectroscope (XPS) demonstrates that as-prepared few-layer graphene has a preserved structure without oxidation or destruction of the sp2 character of the carbon plane. The most important result is that it possesses a higher peroxidase-like catalytic activity than that of GO and RGO. Electrochemical experiments confirmed that the high peroxidase catalytic activity was attributed to the fast electron transfer on the surface of few-layer graphene. Peroxidase activity is appealing and valuable in chemistry, biology [19,20], clinical control [21] and environmental protection [22]. It is frequently used to promote the

* Corresponding author: Fax: +86 592 2183181. E-mail address: [email protected] (J. Weng). 0008-6223/$ - see front matter  2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbon.2013.05.051

52

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degradation of organic toxicants or as a detection tool because of the existence of hydrogen peroxide (H2O2) which is not only a product of biological reactions but also an oxidant in food, pharmaceutical and municipal waste water treatment [23–30]. The precise and rapid detection of H2O2 is of significant importance. However, peroxidase is very expensive, and the enzyme can have a high catalytic ability only in a specified condition. Therefore, it is necessary to find a lowcost and convenient method to use synthetic systems to mimic natural enzymes with high catalytic activity, and it has been a challenge for the last decades. Graphene is easily prepared from graphite with low cost, and it is very stable. At the same time, graphene can serve as the electrical conduction channel for the electron transfer in graphene-based system. Herein, for the first time, exfoliated few-layer graphene is used to determine hydrogen peroxide in three real water samples with satisfactory results. The preparation and application of exfoliated few-layer graphene are low cost and environmentally friendly. These advantages indicate that our present work would provide a new insight into the application of exfoliated few-layer graphene in medicine, biotechnology and environmental chemistry.

2.

Experimental

2.1.

Materials

Chitosan was purchased from Dahao Fine Chemical Products Company (China). Graphite powder was obtained from Tianjin Guangfu Fine Chemical Research Institute (China). H2O2 was purchased from Xilong Chemical (Guangzhou, China). GO and RGO were prepared by the modified Hummers method as carried out in our previous work [7,31,32]. All other reagents are of analytical reagent grade.

2.2.

Preparation of few-layer graphene

Stock solution of chitosan (0.2 mg mL1) was prepared in 0.5% acetic acid (HAc) aqueous solution (pH 4). The graphite powder (1 mg mL1) was dispersed in above solution by sonication for 40 h in a sonic bath (KQ-250 DB). The resulting dispersion was left to stand for 48 h to allow any unstable aggregates to form and then the upper part of dispersion was centrifuged for 10 min at 2000 rpm. After centrifugation, the top of dispersion was decanted by pipet and retained as stock solution. For characterization, part of the suspension was centrifuged at the speed of 12,000 rpm for 30 min to remove the free chitosan, and subjected to 3 cycles of centrifugation (12,000 rpm, 30 min). The final product was redispersed in 0.5% acidic aqueous solution under sonication.

2.3.

Characterization

The micrographs of as-obtained few-layer graphene were taken using a transmission electron microscope (TEM, JEOL JEM-2100) with the accelerating voltage of 200 kV. The samples were prepared by placing a few drops of the colloidal solutions on copper grids coating with lacey carbon film. Atomic force microscopy (AFM) images were acquired in tapping mode in air using a Digital Instrument Nanoscope IIIa.

Thermal gravimetric analysis was performed on a SDT-Q600 instrument under a nitrogen atmosphere at a heating rate of 10 C min1. X-ray diffraction (XRD, Philips PANalytical ˚ ) over the X’Pert) equipped with Cu Ka radiation (k = 1.542 A 2h range of 10–90 was used to characterize the structure of the few-layer graphene. Sample was prepared by depositing a film on the surface of glass slid. Micro-Raman spectroscope (DiLor SA LABRAM) with argon-ion laser at the excitation wavelength of 633 nm, XPS (PHI Quantum 2000) with X-ray source of Mg Ka was used to study the surface composition of the graphene. Xpspeak41 software was used to deconvolute the curves and fit the results. Fourier transform infra-red (FTIR) spectra were recorded on a Nicolet 360 spectrometer (Thermo Scientific), and obtained from KBr pellet samples. Zeta potential analysis was performed using a Malvern Nano-ZS.

2.4.

Peroxidase catalytic activity of few-layer graphene

A series of different concentrations of H2O2 were prepared in aqueous solution (pH = 7). 3,3 0 ,5,5 0 -Tetramethylbenzidine (TMB) was firstly dissolved in dimethyl sulfoxide (4 mM), and then diluted to 100 lM with HAc–sodium acetate buffer (pH 4) [33]. As-obtained few-layer graphene was dispersed in 0.5% acidic aqueous solution (120 lg mL1). Kinetic measurements were carried out in time course mode by monitoring the absorbance at 652 nm on a TU-901 ultraviolet and visible (UV–vis) spectrophotometer. Experiments were carried out by adding 100 lL of TMB (100 lM) and 100 lL of few-layer graphene (120 lg mL1) dispersion to 200 lL of H2O2 with different concentrations. Then the absorption at 652 nm of mixed solution was measured at different times.

2.5.

Electrochemical experiments

Electrochemical experiments were carried out as follows: Gold (Au) electrode was polished with 0.3 and 0.05 lm alumina powder and then washed ultrasonically in 1:1 nitric acid/doubly distilled water and ethanol/doubly distilled water for 2 min. The cleaned Au electrode was dried with nitrogen steam. 10 mg of few-layer graphene, RGO or GO was added to 40 mL of chitosan acetic solution (0.5 mg mL1) to form homogenous dispersion with ultrasonication, respectively. 10 lL of few-layer graphene, RGO or GO dispersion was dropped onto the Au electrode to prepare modified electrodes, respectively. Then the electrodes were dried naturally overnight. Electrochemical measurements were performed on an electrochemical workstation (CHI660C, CH Instrument, USA). The three-electrode system consisted of a platinum wire as auxiliary electrode and an Ag|AgCl (saturated KCl) as reference. Working electrodes were bare or modified gold electrodes. All of the electrochemical experiments were carried out at room temperature and ambient pressure.

2.6.

H2O2 detection in water samples

H2O2 solutions of 30 and 50 lM were prepared with three different water samples – tap water from laboratory, lake water from Furong Lake, and commercial water from supermarket, respectively. No other pretreatment process was performed

CARBON

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before the use of three water samples. Experiments were carried out by adding 100 lL of TMB (100 lM) and 100 lL of as-obtained few-layer graphene (120 lg mL1) dispersion to 200 lL H2O2 solution prepared with three different water samples. Then the absorption at 652 nm of mixed solution was measured at different times. Every experiment was carried out for three times parallelly.

3.

Results and discussion

Graphite powder was firstly dispersed in HAc solution of chitosan. Then the mixture was sonicated for about 40 h (Fig. S1 in Supporting information). Sample for characterization was prepared by standing still, centrifuging, and washing and resuspending. The concentration of the dispersion was 0.123 mg mL1 (Fig. S2 in Supporting information). The yield of few-layer graphene is 12.3% and higher than that of exfoliated few-layer graphene in short time (Table S1 in Supporting information). The absorption coefficient (a) measured at 266 nm was 1678 mL mg1 m1 (Fig. S3 in Supporting information). Chitosan, acting as polymer cationic surfactant, plays a very important role in dispersing few-layer graphene. Therefore, it is necessary to investigate the effect of chitosan concentration on the yield of graphene (details are presented in S1.2 in Supporting information). Fig. S4 indicates that the optimal concentration of chitosan is 0.2 mg mL1. We owed the exfoliation of graphite to the energy provided by the ultrasound wave, which overcomes the van der waals force between graphite layers. Therefore, the ultrasonic time is also an important parameter that should be investigated (details are presented in S1.3 in Supporting information). Fig. S5 demonstrates that the yield of few-layer graphene can be improved with the increasing of ultrosonic time. In our experiment, the appropriate ultrosonic time is 40 h. The resulted suspension of few-layer graphene has a good stability in a certain range of pH from 1.5 to 5 (Fig. S6 in Supporting information). It also has good pH-responsive ability and can be switched reversibly between a well dispersed and a more aggregated state by adjusting pH from 4 to 9 (Fig. S6b in Supporting information). Zeta potential in Fig. S6c further confirms the good redispersibility of few-layer graphene while adjusting pH from 9 to 4. The dispersing mechanism of graphene with the aid of chitosan is presented in Fig. S7. Fig. S8 in Supporting information further demonstrates that the function of chitosan in preparing few-layer graphene.

3.1.

Structure characterization

TEM is frequently used to character the structure and border patterns of graphene or few-layer graphene [34–36]. Here, a detailed TEM analysis was carried out to investigate the exact form of as-obtained few-layer graphene in the dispersions (Fig. 1 and Fig. S9 in Supporting information). We estimate the layer number (n) per flake by examining the edges of the flakes. The edges of multilayers graphene in TEM images are always distinguishable. The images in inset were obtained with high resolution, the dark lines near the edge indicate the thickness of graphene or few-layer graphene. Few-layer or multilayer graphene (Fig. 1a and b), graphene (Fig. 1c and d)

53

and folded graphene (Fig. S9b), can be found in the sample. These graphenes have the sizes of several hundred nanometers. Some restackable sheets can also be observed (Fig. S9a and c in Supporting information), which is easy to understand because graphene is thermodynamically unstable in theory and easily clings to crimp and restacks with each other to lower free energy [37]. Graphite cannot be exfoliated after sonication of 40 h without chitosan in water (Fig. S9d in Supporting information). Therefore, chitosan plays a very important role in exfoliation of graphite. AFM was used to further investigate the structure of fewlayer graphene (Fig. 2). The monolayer graphene (Fig. 2A) with the height of 0.997 nm is consistent with publications showing the apparent height (1 nm) of graphene monolayers [14]. It is higher than the theoretical thickness (0.334 nm) of monolayer graphene. This result may be due to the adsorbed chitosan molecules on two surfaces of graphene, which will be confirmed by XPS and FTIR in latter paragraphs. At the same time, few-layer or multilayer graphene (Fig. 2B–D) is also observed. Therefore, it is necessary to further improve the yield of monolayer graphene in the future. XRD is also a significant technology to characterize the structure of graphene. Fig. 3 demonstrates that graphite has a perfect characteristic peak at 2h = 26.5, corresponding to a typical inter-spacing of 0.334 nm. Two weaker peaks at 2h = 20.1 and 10 for chitosan is also observed. In contrast, there is not strong characteristic peak for few-layer graphene, which was similar to RGO [38–40]. It is easy to understand in that the ordered structure of pristine graphite has been destroyed after exfoliation. The broad and weak band may be attributed to the residual chitosan and few-layer graphene sheets. It is significant to characterize the quality of the few-layer graphene. The presence of defects such as oxides has a bad effect on the electronic properties of graphene. Raman and XPS techniques were used to determine whether the exfoliation process would lead to the formation of defects. Fig. 4 exhibits Raman spectra of graphite and as-obtained few-layer graphene. Generally three bands are of characterization: Dband (1350 cm1), G-band (1582 cm1), and 2D-band (2700 cm1). The D band is the characterization of defect caused by edge effects or topological defects in the sheets [14,41]. We note that the defect can hardly be observed in the pristine graphite powder but visible in as-obtained fewlayer graphene. We attribute this to the ultrasonic treatment that makes graphite exfoliate but decrease in flake dimensions, just as the TEM characterization where we can see most of few-layer graphene are small flakes (from 200 to 500 nm) with sharp edges. The 2D band is also an evidence of exfoliation. In contrast to pristine graphite, the 2D-band of few-layer graphene is broadened and red-shifted, and the important information that the intensity of 2D peak in few-layer graphene increases greatly (after normalizing G band intensity) is an obvious evidence to confirm that graphite has been exfoliated after ultrasonication [13,14]. However, compared with the intensity of D peak in the spectrum of few-layer graphene, the weaker intensity of 2D peak demonstrates that as-obtained graphene is few-layer or multilayer [42], which is in consistent with TEM and AFM.

54

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Fig. 1 – TEM images of graphene with different layers. (a) n P 10; (b) 2 6 n 6 5; (c) and (d) n = 1 (n: number of layer). Insets in (a–c) are the images with high resolution.

XPS is of interest in the present study because of the inherently high sensitivity of this technique to the valence state of the surface element. Therefore, XPS was selected to analyze the surface groups of few-layer graphene. Fig. 5 presents the XPS survey scan spectra of graphite and as-obtained fewlayer graphene. Obviously, the value of IC1s/IO1s (I-intensity) of graphite is larger than that of few-layer graphene. The increasing content of oxygen element in as-obtained fewlayer graphene may be resulted from the adsorbed chitosan on the surface because chitosan contains C–O, C@O (part of acetylated unit) and C–N bonds. Therefore, some fitting procedures are necessary to deconvolute the C 1s peak. Curve fitting of the C 1s spectrum was carried out by a Gaussian– Lorentzian peak shape after performing a Shirley background correction. The C 1s signal of few-layer graphene exhibits four different peaks (Fig. 5c): C–C (284.8 eV), C–N (285.6 eV), C–O (286.6 eV) and C@O (287.9 eV) [43,44], which indicate that there may be some residual chitosan molecules on the surface of few-layer graphene. The N 1s peak at 400 eV also confirms the existence of adsorbed chitosan on the surface of graphene. The FT-IR spectra of as-obtained few-layer graphene and chitosan further demonstrate that some chitosan molecules are residual molecules (Fig. S10 in Supporting information). At the same time, the content of C@O is negligible because the content of acetylated unit in chitosan is very low, which further demonstrates that the defect resulted from oxidation could be neglected. According to analysis of element content, the calculated content of chitosan on

few-layer graphene is about 15%. All of the characterizations above indicate that we have successfully prepared few-layer graphene by simply ultrasound treatment in the presence of chitosan, however, further study is needed to carry out to improve the yield of graphene.

3.2.

Peroxidase catalytic activity of few-layer graphene

Peroxidase is an effective catalyst for the detection of H2O2 in biosensor [24,45]. However, some problems must be noticed: peroxidase is very expensive, and the enzyme can have a high catalytic ability only in a specified condition. Therefore, it is necessary to find a low-cost and convenient method. In this study, few-layer graphene prepared by our method has a higher peroxidase catalytic ability in the decomposition of H2O2 than that of GO and RGO. This process is realized in the presence of TMB which will change its color from achromatous to blue during the decomposition reaction (Fig. 6b insert). TMB is a kind of derivative of benzidine. It has been frequently used as a chromogenic peroxidase substrate. The TMB cation free radical, a one-electron oxidation product, would be formed with exposure of TMB to peroxidase and H2O2. It is responsible for the blue color (maximum absorbance at 652 nm) that develops during TMB oxidation. Further oxidation with peroxidase and H2O2 yields the diimine (maximum absorbance at 450 nm), a yellow two-electron oxidation product [23,24,46,47]. Therefore, the oxidation product of TMB

CARBON

A

B

3

nm

2

nm

55

6 2 (2 0 13 ) 5 1–60

0

0

-2

-3 0

50

nm

100

150

200

65

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195

260

325

nm

C

D

6

nm

nm

10 0

0

-6 150

300

450

600

200

400

nm

nm

600

800

Fig. 2 – AFM images of graphene and few-layer graphene. (A) monolayer, (B) three layers and (C and D) multilayers. Bottom of each image is the height profile along the green line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

50k

2.4k

40k

1.2k

Chitosan

20k

Few-layer graphene

10k

Intensity

Intensity

1.8k 30k

Intensity

Graphite

Few-layer graphene

600.0 Graphite

0

0.0 0

20

40

60

80

2θ

Fig. 3 – XRD patterns of graphite, chitosan and few-layer graphene, respectively.

1200

1600

2000

2400

2800

Wavenumber ( cm-1)

Fig. 4 – Raman spectra of as-obtained few-layer graphene and graphite.

56

CARBON

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6 2 ( 2 0 1 3 ) 5 1 –6 0

(b)

Graphite

Few-layer graphene

C 1s

0

200

400

600

800

1000

1200

0

200

Binding Energy (eV)

400

O 1s

600

800

1000

Binding Energy (eV)

(d)

(c)

N 1s

C 1s C-C C-O C=O C-N

294

291

288

285

282

410

405

Binding Energy (eV)

400 395 Binding energy (eV)

390

0.6 0.5

(b) 0.4

time

Absorbance at 652 nm

(a) Absorbance at 266 nm

Fig. 5 – Survey scan spectra of graphite (a) and few-layer graphene (b). C 1s (c) and N 1s (d) XPS spectra of few-layer graphene.

0.4 0.3 0.2 0.1

3

0.3

0.2

0.1

2 1

0.0

0.0 400

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Wavelength (nm)

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Time(s)

H2O2 (μΜ)

50 40

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30 20

0.2 10

0.1

5 1 0.5 0.05

0.0 0

500

1000

Time (s)

1500

2000

Absorbance at 652 nm

(d) 0.3

(c) 0.4 Absorbance at 652 nm

200

0.2

0.1

0.0 0

10

20

30

40

50

H2O2 (μΜ)

Fig. 6 – (a) UV–vis spectra for the mixed solution of TMB and H2O2 obtained at same intervals after the addition of 30 lg mL1 as-obtained few-layer graphene at room temperature in pH 4 solution. (b) The time-dependent absorbance changes at 652 nm of 400 lL TMB (25 lM) reaction solutions: (1) 30 lg mL1 of few-layer graphene; (2) 0.1 mM H2O2; (3) 30 lg mL1 fewlayer graphene and 0.1 mM H2O2. Inset is the photograph of above three solutions after 10 min. (c) The timedependent absorbance changes at 652 nm with different concentrations of H2O2 at room temperature. (d) Linear calibration plot for H2O2.

agent, GO can be transformed into RGO [7,31]. During this process some of the defect can be removed while large defect populations remain, which continue to disrupt the electronic performances. Therefore, graphene with little defect has a higher conductivity than that of RGO and GO, which results in a high catalytic activity. In order to further understand the high catalytic activity of exfoliated few-layer graphene, the electrochemical properties of as-obtained few-layer graphene, RGO and GO were investigated in commonly electrochemical reagent, K3Fe(CN)6. Fig. 8 plots the cyclic voltammograms (CVs) of bare gold (Au), few-layer graphene-, RGOand GO-modified Au electrodes in 5 mM K3Fe(CN)6 and 1.0 M KCl aqueous solution. Obviously, few-layer graphene-modified electrode has the highest current intensity than that of bare Au, RGO- and GO-modified, which indicates the high conductivity of as-obtained few-layer graphene. At the same time, the peak-to-peak separation (DEp) at the few-layer graphene, bare Au, RGO and GO are 71, 77, 101 and 104 mV, respectively, which demonstrates that as-obtained few-layer graphene would accelerate the electron transfer between K3Fe(CN)6 and electrode [53,54]. To further investigate the electrochemical properties of asobtained few-layer graphene, RGO and GO, the CVs of modified-electrodes in 5 mM K3Fe(CN)6 with different scan rates

60

Few-layer graphene 40

Bare Au

(

has two strong absorption peaks at 450 and 652 nm. Recording the absorbance at 652 nm was used to monitor the progress or kinetics of the reaction because the peak at 652 nm was much stronger than that at 450 nm (Fig. 6a). Fig. S11 in Supporting information demonstrates that the reaction rate increases with the few-layer graphene concentration increasing to 30 lg mL–1. The stability of few-layer graphene depends on the pH of the dispersion and the optimal pH is nearly 4 (Fig. S6a in Supporting information). Research as to the catalytic activity of few-layer graphene from 22 to 52 C indicated that the optimal temperature was 42 C (Fig. S12 in Supporting information). The optimal pH and temperature are similar to the values reported for horseradish peroxidase (HRP) [23,24]. Fig. 6b demonstrates that both of H2O2 and as-obtained few-layer graphene cannot alone oxidize TMB efficiently. Therefore, TMB oxidation is resulted from the decomposition of H2O2 by as-obtained few-layer graphene. The concentration response curve of H2O2 to the absorbance of TMB is presented in Fig. 6c. A linear relationship is established in the range of 0.05–50 lM with a correlation coefficient of 0.9942. The regression equation is absorbance = 0.005 C + 0.0257 (C is the concentration of H2O2 in lM) with a detection limit of 10 nM (Fig. 6d), which is lower than that of Se/Pt composite (3.1 lM) [48], Au–graphene–HRP (1.7 lM) [22], graphene and ZnO composites-based amperometric biosensor (0.6 lM) [49], magnetite–graphene-based biosensor (0.5 lM) [50], and graphene-modified electrode (0.11 lM) [51]. We also compared the catalytic activity of few-layer graphene with RGO and GO prepared by our group (Fig. 7) within 600 s. The result indicates that graphene has better catalytic activity than that of GO and RGO, and the peroxidase catalytic activity of few-layer graphene is 45 times higher than that of GO and 4 times higher than that of RGO with the same concentration of 30 lg mL1. The high catalytic activity of the as-obtained few-layer graphene is attributed to its high conductivity because it was directly exfoliated from graphite by chitosan and had lower defect than that of RGO and GO. During the preparation of GO, a considerable amount of surface oxygen groups such as epoxides, hydroxides, and carboxylic groups were introduced into the graphene, resulting a significant loss of electrical conductivity of GO [8,10,52]. Under the action of reducing

RGO

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60

0.2 -1

30 μg mL RGO

0.1

0.1

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Fig. 8 – CVs of bare Au, few-layer graphene-, RGO- and GOmodified Au electrodes in 5 mM K3Fe(CN)6 and 1.0 M KCl aqueous solution (scan rate 0.1 V s1).

30 μg mL-1 Few-layer graphene

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120

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Absorbance at 652 nm

57

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Current ( μΑ

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0 -60

Few-layer graphene without H2O2 -120 -180

-1

30 μg mL GO

-240

0.0 0

100

200

300

400

500

600

Time (s)

Fig. 7 – Comparison of the peroxidase catalytic activity of few-layer graphene, GO and RGO. The reaction solution contains 25 lM TMB and 0.1 mM H2O2.

-0.1

0.0

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0.3

0.4

0.5

0.6

Potential (V)

Fig. 9 – CVs of bare Au, few-layer graphene-, RGO- and GOmodified electrodes in N2-saturated phosphate buffer (pH 7.4) containing 5 mM H2O2 at the scan rate of 0.3 V s1.

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Table 1 – Summary of the reaction rate constant (k), activation energy (Ea), pre-exponential factors (A), and the entropy of activation (DS). T (K)

1000/T (K1)

k (min1)

Ea (kJ mol1)

A (min1)

DS (J mol1 K1)

298.15 303.15 308.15

3.35 3.3 3.24

0.106 ± 0.04 0.125 ± 0.035 0.155 ± 0.042

28.8

1.15 · 104

77.8

Table 2 – Determination of H2O2 in water samples (n = 3). Sample

Added (lM)

Detection (lM)

Recovery (%)

RSD (%)

Tap water

15 25 15 25 15 25

15.32 25.60 14.92 25.06 14.86 24.86

102.1 102.4 99.5 100.2 99.1 99.4

0.8 2.0 1.7 0.7 1.7 2.4

Commercial water Lake water

from 0.05 to 5 V s1 are exhibited in Fig. S13a, c and e. Both of anodic and cathodic peak currents increased linearly with the square root of scan rate increasing (Fig. 13b, d and f). Based on these CV datas and formula (1) [53] ip =A ¼ ð2:69  105 Þ  n3=2 

D01=2

 C0  v1=2

ð1Þ

where ip is the peak current, the electron in the electrode reaction, n = 1, the diffusion coefficient of K3Fe(CN)6, D0 = 0.76 · 105 cm2 s1, the concentration of K3Fe(CN)6, C0 = 5 mM, t is the scan rate. The calculated electrochemically active surface area (ESA) of few-layer graphene (0.038 cm2) is nearly 2.4 times higher than that of RGO (0.016 cm2) and 3 times higher than that of GO (0.012 cm2). The high ESA of few-layer graphene also indicates that it would have higher conductivity than that of RGO and GO. Besides K3Fe(CN)6, we further investigated the electrocatalytic activity of H2O2 by as-obtained few-layer graphene, RGO and GO to understand the high catalytic activity of few-layer graphene. Fig. 9 demonstrates the electrocatalysis toward H2O2 at different electrodes. Obviously, few-layer graphenemodified electrode has a higher current intensity, which indicates that it has a higher catalytic activity toward H2O2 than that of RGO and GO. Fig. S14a, c and e present the CVs of three different electrodes in H2O2 with different scan rates from 0.1 to 3 V s1. The oxidative peak currents increased linearly with the square root of scan rate increasing (Fig. S14b, d and f). By calculating ratio of the slope of current vs square root of scan rate, the ESA of few-layer graphene is about 2 times higher than that of RGO and 2.3 times higher than that of GO, which further confirms that few-layer graphene has higher conductivity than that of RGO and GO. The kinetics and thermodynamics of the catalytic reaction of H2O2 by few-layer graphene were further studied in a range of 25–35 C. The pseudo-first-order kinetics (Fig. S15 in Supporting information) with respect to the oxidation of TMB can be applied to our experimental system (Eq. (2)). y ¼ y0 þ y1 ekt

ð2Þ

Fitting the absorbance data of three different temperatures to Eq. (2) yields the reaction rate constant k (three independent

measurements were carried out for each temperature). The rate constant increases with increasing temperature (Table 1). In addition, based on the Arrhenius Eq. (3): ln k ¼ ln A  Ea=RT

ð3Þ

the apparent activation energy (Ea) of the H2O2 decomposition in the presence of graphene is 28.8 kJ mol1 (Table 1) which is lower than that of other H2O2 catalyst system, such as iron oxide (60 kJ mol1) [55], bimetallic Pt–Pd (36.4 kJ mol1) [56], ferrihydrites (76.13 kJ mol1) [57], indicating a high catalytic activity for the as-prepared few-layer graphene. In addition to Ea, the pre-exponential factor (A) can also be obtained from the intercept of the linear dependence of ln(1/ k) vs 1000/T, and the entropy of activation (DS) can be received from Eq. (4) [58]. These datas are presented in Table 1. ln A ¼ DS=R

ð4Þ

The rapid and accurate determination of hydrogen peroxide is of considerable interest to researchers because of its significance in environmental, clinical, food, and pharmaceutical analyses [34]. Here as-obtained few-layer graphene was used to determine hydrogen peroxide concentration in three real water samples. Three parallel experiments were performed for each measurement. Standard addition method was carried out to confirm the feasibility of the system. Satisfactory results were received and presented in Table 2. The recoveries were in the range of 100.1–102.8%, and the relative standard deviations were less than 3%.

4.

Conclusions

We have demonstrated a simple method to prepare pristine few-layer graphene in aqueous solution in the presence of chitosan. The chemical structure and morphology were fully characterized by TEM, AFM, XRD, XPS, FT-IR, Raman and UV–vis spectroscopies. The most important discovery is that as-obtained few-layer graphene has a higher peroxidase-like activity than that of GO and RGO, which indicates that our present work would provide a new insight into the application of exfoliated graphene or few-layer graphene. The high catalytic activity of few-layer graphene is attributed to its high

CARBON

6 2 (2 0 13 ) 5 1–60

conductivity confirmed by electrochemical technique. The kinetics and thermodynamics of the catalytic reaction were further studied. The activation energy is about 28.8 kJ mol1, the entropy of activation is 77.8 J mol1 K1 and the pre-exponential factor is 1.15 · 104 min1. At last the as-prepared fewlayer graphene was used to determine hydrogen peroxide concentration in three real water samples with satisfactory results. In general, the activity of natural enzyme is easy to lost in a severe environment, such as high temperature. However, as-prepared few-layer graphene as a mimic peroxidase is thermostabile and easily prepared. All these advantages provide it a potential application in clinic diagnostics and environmental analysis.

[10]

[11] [12]

[13]

[14]

Acknowledgements

[15]

This work is supported by Natural Science Foundation of China (No. 81171453), the Fundamental Research Funds for the Central Universities, Program for New Century Excellent Talents in University, the Ministry of Education, and Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

[16]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbon. 2013.05.051.

[17]

[18]

[19]

[20]

[21] R E F E R E N C E S

[22] [1] Novoselov KS, Geim AK, Morozov SV, Jiang D, Zhang Y, Dubonos SV, et al. Electric field effect in atomically thin carbon films. Science 2004;306(5696):666–9. [2] Stoller MD, Park S, Zhu Y, An J, Ruoff RS. Graphene – based ultracapacitors. Nano Lett 2008;8(10):3498–502. [3] Geim AK, Novoselov KS. The rise of graphene. Nat Mater 2007;6(3):183–91. [4] Tang Z, Wu H, Cort JR, Buchko GW, Zhang Y, Shao Y, et al. Constraint of DNA on functionalized graphene improves its biostability and specificity. Small 2010;6(11):1205–9. [5] Chung C, Kim YK, Shin D, Ryoo SR, Hong BH, Min DH. Biomedical applications of graphene and graphene oxide. Acc Chem Res 2013. http://dx.doi.org/10.1021/ar300159f, on line. [6] Wen H, Dong C, Dong H, Shen A, Xia W, Cai X, et al. Engineered redox-responsive PEG detachment mechanism in PEGylated nano-graphene oxide for intracellular drug delivery. Small 2012;8(5):760–9. [7] Jia J, Sun L, Hu N, Huang G, Weng J. Graphene enhances the specificity of the polymerase chain reaction. Small 2012;8(13):2011–5. [8] Stankovich S, Dikin DA, Piner RD, Kohlhaas KA, Kleinhammes A, Jia Y, et al. Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carbon 2007;45(7):1558–65. [9] Stankovich S, Piner RD, Chen X, Wu N, Nguyen ST, Ruoff RS. Stable aqueous dispersions of graphitic nanoplatelets via the reduction of exfoliated graphite oxide in the presence of

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

59

poly(sodium 4-styrenesulfonate). J Mater Chem 2006;16(2):155–8. Bai H, Li C, Shi G. Functional composite materials based on chemically converted graphene. Adv Mater 2011;23(9):1089–115. Park S, Ruoff RS. Chemical methods for the production of graphenes. Nat Nanotechnol 2009;4(4):217–24. Eda G, Fanchini G, Chhowalla M. Large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material. Nat Nanotechnol 2008;3(5):270–4. Hernandez Y, Nicolosi V, Lotya M, Blighe FM, Sun Z, De S, et al. High-yield production of graphene by liquid-phase exfoliation of graphite. Nat Nanotechnol 2008;3(9):563–8. Lotya M, Hernandez Y, King PJ, Smith RJ, Nicolosi V, Karlsson LS, et al. Liquid phase production of graphene by exfoliation of graphite in surfactant/water solutions. J Am Chem Soc 2009;131(10):3611–20. Bao H, Pan Y, Ping Y, Sahoo NG, Wu T, Li L, et al. Chitosanfunctionalized graphene oxide as a nanocarrier for drug and gene delivery. Small 2011;7(11):1569–78. Feng W, Li Y, Ji P. Interaction of water soluble chitosan with multiwalled carbon nanotubes. AIChE J 2012;58(1):285–91. Iamsamai C, Hannongbua S, Ruktanonchai U, Soottitantawat A, Dubas ST. The effect of the degree of deacetylation of chitosan on its dispersion of carbon nanotubes. Carbon 2010;48(1):25–30. Yan LY, Poon YF, Chan-Park MB, Chen Y, Zhang Q. Individually dispersing single-walled carbon nanotubes with novel neutral pH water-soluble chitosan derivatives. J Phys Chem C 2008;112(20):7579–87. Jv Y, Li B, Cao R. Positively-charged gold nanoparticles as peroxidase mimic and their application in hydrogen peroxide and glucose detection. Chem Commun 2010;46(42):8017–9. Cui X, Liu G, Lin Y. Biosensors based on carbon nanotubes/ nickel hexacyanoferrate/glucose oxidase nanocomposites. J Biomed Nanotechnol 2005;1(3):320–7. Lu X, Zhou J, Lu W, Liu Q, Li J. Carbon nanofiber-based composites for the construction of mediator-free biosensors. Biosens Bioelectron 2008;23(8):1236–43. Zhou K, Zhu Y, Yang X, Luo J, Li C, Luan S. A novel hydrogen peroxide biosensor based on Au–graphene–HRP–chitosan biocomposites. Electrochim Acta 2010;55(9):3055–60. Gao L, Zhuang J, Nie L, Zhang J, Zhang Y, Gu N, et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat Nanotechnol 2007;2(9):577–83. Song Y, Qu K, Zhao C, Ren J, Qu X. Graphene oxide: intrinsic peroxidase catalytic activity and its application to glucose detection. Adv Mater 2010;22(19):2206–10. Liu R, Li S, Yu X, Zhang G, Zhang S, Yao J, et al. Facile synthesis of Au-nanoparticle/polyoxometalate/graphene tricomponent nanohybrids: an enzyme-free electrochemical biosensor for hydrogen peroxide. Small 2012;8(9):1398–406. Cao X, Zeng Z, Shi W, Yep P, Yan Q, Zhang H. Threedimensional graphene network composites for detection of hydrogen peroxide. Small 2013;9(10):1703–7. Xu F, Sun Y, Zhang Y, Shi Y, Wen Z, Li Z. Graphene–Pt nanocomposite for nonenzymatic detection of hydrogen peroxide with enhanced sensitivity. Electrochem Commun 2011;13(10):1131–4. Wang J, Lin Y, Chen L. Organic-phase biosensors for monitoring phenol and hydrogen peroxide in pharmaceutical antibacterial products. Analyst 1993;118(3):277–80. Shu X, Chen Y, Yuan H, Gao S, Xiao D. H2O2 sensor based on the room-temperature phosphorescence of Nano TiO2/SiO2 composite. Anal Chem 2007;79(10):3695–702. Huang KJ, Niu DJ, Liu X, Wu ZW, Fan Y, Chang YF, et al. Direct electrochemistry of catalase at amine-functionalized

60

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

CARBON

6 2 ( 2 0 1 3 ) 5 1 –6 0

graphene/gold nanoparticles composite film for hydrogen peroxide sensor. Electrochim Acta 2011;56(7):2947–53. Dong J, Weng J, Dai LZ. The effect of graphene on the lower critical solution temperature of poly(N-isopropylacrylamide). Carbon 2013;52:326–36. Yang Q, Wang Z, Weng J. Self-assembly of natural tripeptide glutathione triggered by graphene oxide. Soft Matter 2012;8(38):9855–63. Bos E, Van der Doelen A, Rooy Nv, Schuurs A. 3,3 0 ,5,5 0 Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J Immunoassay Immunochem 1981;2(3–4):187–204. Dato A, Radmilovic V, Lee Z, Phillips J, Frenklach M. Substrate-free gas-phase synthesis of graphene sheets. Nano Lett 2008;8(7):2012–6. Shen Z, Li J, Yi M, Zhang X, Ma S. Preparation of graphene by jet cavitation. Nanotechnology 2011;22(36). 365306(7pp). Reina A, Jia X, Ho J, Nezich D, Son H, Bulovic V, et al. Large area, few-layer graphene films on arbitrary substrates by chemical vapor deposition. Nano Lett 2008;9(1):30–5. Meyer JC, Geim AK, Katsnelson MI, Novoselov KS, Booth TJ, Roth S. The structure of suspended graphene sheets. Nature 2007;446(7131):60–3. Guo HL, Wang XF, Qian QY, Wang FB, Xia XH. A green approach to the synthesis of graphene nanosheets. ACS Nano 2009;3(9):2653–9. Chen W, Yan L, Bangal PR. Preparation of graphene by the rapid and mild thermal reduction of graphene oxide induced by microwaves. Carbon 2010;48(4):1146–52. Zhu Y, Stoller MD, Cai W, Velamakanni A, Piner RD, Chen D, et al. Exfoliation of graphite oxide in propylene carbonate and thermal reduction of the resulting graphene oxide platelets. ACS Nano 2010;4(2):1227–33. Khan U, O’Neill A, Lotya M, De S, Coleman JN. Highconcentration solvent exfoliation of graphene. Small 2010;6(7):864–71. Ferrari AC, Meyer JC, Scardaci V, Casiraghi C, Lazzeri M, Mauri F, et al. Raman spectrum of graphene and graphene layers. Phys Rev Lett 2006;97(18):187401(4pp). Park OK, Hahm MG, Lee S, Joh HI, Na SI, Vajtai R, et al. In situ synthesis of thermochemically reduced graphene oxide conducting composites. Nano Lett 2012;12(4):1789–93. Liu J, Fu S, Yuan B, Li Y, Deng Z. Toward a universal ‘‘adhesive nanosheet’’ for the assembly of multiple nanoparticles based on a protein-induced reduction/decoration of graphene oxide. J Am Chem Soc 2010;132(21):7279–81. Sanz V, De Marcos S, Castillo JR, Galba´n J. Application of molecular absorption properties of horseradish peroxidase

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

for self-indicating enzymatic interactions and analytical methods. J Am Chem Soc 2005;127(3):1038–48. Josephy PD, Eling T, Mason RP. The horseradish peroxidasecatalyzed oxidation of 3,5,3 0 ,5 0 -tetramethylbenzidine. Free radical and charge–transfer complex intermediates. J Biol Chem 1982;257(7):3669–75. Rhee SG, Chang TS, Jeong W, Kang D. Methods for detection and measurement of hydrogen peroxide inside and outside of cells. Mol Cells 2010;29(6):539–49. Li Y, Zhang JJ, Xuan J, Jiang LP, Zhu JJ. Fabrication of a novel nonenzymatic hydrogen peroxide sensor based on Se/Pt composites. Electrochem Commun 2010;12(6):777–80. Xu J, Liu C, Wu Z. Direct electrochemistry and enhanced electrocatalytic activity of hemoglobin entrapped in graphene and ZnO nanosphere composite film. Microchim Acta 2010;172(3–4):425–30. He Y, Sheng Q, Zheng J, Wang M, Liu B. Magnetite–graphene for the direct electrochemistry of hemoglobin and its biosensing application. Electrochim Acta 2011;56(5):2471–6. Li M, Xu S, Tang M, Liu L, Gao F, Wang Y. Direct electrochemistry of horseradish peroxidase on graphenemodified electrode for electrocatalytic reduction towards H2O2. Electrochim Acta 2011;56(3):1144–9. Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, et al. Graphene and graphene oxide: synthesis, properties, and applications. Adv Mater 2010;22(35):3906–24. Weng J, Xue J, Wang J, Ye J, Cui H, Sheu F, et al. Gold-cluster sensors formed electrochemically at boron-doped-diamond electrodes: detection of dopamine in the presence of ascorbic acid and thiols. Adv Funct Mater 2005;15(4):639–47. Li XR, Kong FY, Liu J, Liang TM, Xu JJ, Chen HY. Synthesis of potassium-modified graphene and its application in nitriteselective sensing. Adv Funct Mater 2012;22(9):1981–8. Huang C, Huang Y, Cheng H, Huang Y. Kinetic study of an immobilized iron oxide for catalytic degradation of azo dye reactive black B with catalytic decomposition of hydrogen peroxide. Catal Commun 2009;10(5):561–6. Hasnat MA, Rahman MM, Borhanuddin SM, Siddiqua A, Bahadur NM, Karim MR. Efficient hydrogen peroxide decomposition on bimetallic Pt–Pd surfaces. Catal Commun 2010;12(4):286–91. Ma Y, Meng S, Qin M, Liu H, Wei Y. New insight on kinetics of catalytic decomposition of hydrogen peroxide on ferrihydrite: based on the preparation procedures of ferrihydrite. J Phys Chem Solids 2012;73(1):30–4. Narayanan R, El-Sayed MA. Shape-dependent catalytic activity of platinum nanoparticles in colloidal solution. Nano Lett 2004;4(7):1343–8.